PROJECT SUMMARY In this application, we will characterize CD4 T cells reactive to epitopes of the fungal allergens Aspergillus fumigatus (ASP) and Alternaria alternata (ALT) to understand the molecular basis of severe asthma. Sensitization and/or exposure to antigens (spores) from fungal species such as ASP and ALT have been strongly associated with risk for a number of asthma-severity-related adverse outcomes. Based on this premise, we will test the hypotheses that (i) Fungal allergen-specific CD4 T cells are more pathogenic and qualitatively different when compared to allergen-specific CD4 T cells directed towards non-fungal inhaled allergens such as house dust mite (HDM), cockroach (CR), mouse (MO) or potential oral allergens such as cow milk (CM). (ii) Fungal allergen-epitope-specific CD4 T cells in patients with severe asthma have more pathogenic features than those from mild asthmatics. We will capitalize on subjects enrolled in three large asthma cohorts for these studies. In Aim 1, we will define epitopes recognized by Aspergillus (ASP) and Alternaria (ALT)-sensitized asthmatics categorized based on disease severity. The reactivity of predicted ASP and ALT epitopes will be tested in 20 sensitized subjects with mild and severe asthma and in 20 non-sensitized subjects without asthma to assess the nature of allergic versus healthy responses. By comparing the epitope-reactivity of the three cohorts, we will define epitopes that are common or specific to each subgroup: severe asthma, mild asthma and non- sensitized non-asthmatics. In Aim 2, we will determine the molecular profile of fungal (ASP and ALT)-specific CD4 T cells in patients with different asthma severity. We will utilize fungal epitope mega-pools to define the phenotype and transcriptional profile (at bulk and single-cell level) of ASP and ALT epitope-specific CD4 T cell responses in 10-20 fungal-sensitized subjects with severe and mild-to-moderate asthma, allergic bronchopulmonary aspergillosis (ABPA) and in control subjects without asthma. In parallel, we will also assess the molecular profile of fungal-specific CD4 T cells present in the airways of asthmatic subjects. To determine if the molecular features of fungal allergen-specific CD4 T cells are different from allergen-specific CD4 T cells directed to HDM, CR, MO, CM or antigen-specific Th2 cells induced by vaccination (pertussis and tetanus), we will perform additional transcriptomic analysis of these cells, and through the integrated Data Management Core specializing in bioinformatics cross compare data sets with Project 1 and 2. Together these fungal epitope validation studies in subjects with varying asthma severity will provide insights into the nature of CD4 T cell responses that drive pathogenesis of severe asthma.